Our understanding of how Ras proteins function as molecular switches that relay signals initiated at the cell surface by extracellular stimuli that control cell growth and differentiation has increased considerably during the past year. Both the key regulators of Ras activity (GAPs and GEFs) and the essential downstream components of the Ras signal transduction pathway have now been identified. However, while a detailed picture of Ras-mediated signaling has now been defined, the precise role of each component and how the information is relayed from component to component remains to be elucidated. The overall goals of this proposal are (1) to determine the role of upstream activators (GEFs) and downstream mediators of Ras signal transduction in Ras-mediated transformation, and (2) to determine whether two Ras-related proteins (TC21 and R-Ras) also utilize these same components for signal transduction and for triggering malignant transformation. The specific aims of this proposal are (1) to determine if Ras GEFs (CDC25 and SOS1) are also activators of other members of the Ras superfamily of proteins, (2) to establish the role of the mitogen-activated protein (MAP kinase cascade of serine/threonine kinases (MEKs and MAPKs) in Ras transformation, (3) to establish the role of the Ras-related TC21/R-RasB protein in human carcinogenesis, (4) to establish whether the Ras-related R-Ras protein is functionally distinct from TC21 and whether its activities are regulated by Bcl-2, and (5) to determine if CAAX-based peptidomimetics are specific inhibitors of Ras prenylation and transformation, and whether they also function as inhibitors of TC21 and R-Ras transformation. Our increased understanding of how Ras proteins mediate signaling pathways that control normal cell growth and differentiation, and how oncogenic Ras proteins perturb these pathways, will have two important implications for cancer biology. First, the deregulated function of other components of the Ras signal transduction pathway (e.g., Ras GEFs), in the absence of Ras mutations, may also be important in cancer. Second, any essential components of Ras signaling (e.g., the MEK/MAPK serine/threonine kinases) may represent novel targets for rational drug design. The studies that we have proposed will provide further insight into both possibilities.